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Recent evidence suggests that Gag targeting to late endosomal\/multivesicular body (LE\/MVB) compartments occurs prior to viral particle budding at the plasma membrane (PM). However, the route that Gag follows before reaching its steady-state destinations still remains a subject of debate. Using a subcellular fractionation method that separates PM from LE\/MVB combined with pulse-chase labeling, we analyzed Gag trafficking in HIV-1-producing HEK 293T cells. Our results reveal that the majority of newly synthesized Gag is primarily targeted to the PM. While PM-targeted Gag was efficiently released, a significant fraction of the remaining cell surface-associated Gag was found to be subsequently internalized to LE\/MVB, where it accumulated, thus accounting for the majority of LE\/MVB-associated Gag. Importantly, this accumulation of Gag in LE\/MVB was found to be cholesterol dependent since it was sensitive to the sterol-binding drugs filipin and methyl-\u03b2-cyclodextrin. 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